What is indirect immunofluorescence?

Indirect immunofluorescence, or secondary immunofluorescence, is a technique used in laboratories to detect circulating autoantibodies in patient serum. It is used to diagnose autoimmuneblistering diseases.

Unlabelled primary antibodies from the patient serum bind to the target molecule in pre-prepared tissue samples.

Secondary antibodies, conjugated with a fluorescent dye such as fluorescein isothiocyanate (FITC), bind to the primary antibody.

When exposed to light, the fluorescent dye is excited and emits a wavelength that can be seen with a fluorescent microscope.

The tissue substrates used (monkey oesophagus or human skin) will depend on the suspected disease. Different staining patterns will help diagnose an autoimmunebullous disease.

Describe the laboratory method

Preparing patient serum for testing

The blood sample is then centrifuged to separate the serum from the clot.

Serum is aliquoted and frozen until testing.

Indirect immunofluorescence test

Prepare 4–6 micron thick slices of tissue substrate slides (this can be done in a laboratory or obtained commercially)

Several slides are prepared with different tissue substrates.

Tissue section is incubated with patient serum for 30 minutes and doubling dilution is performed if a titre is required.

The slides are washed to remove any unbound primary antibodies.

Slides are incubated for another 30 minutes with secondary antibodies containing fluorescent dye.

The slide is mounted under a cover slip.

It is examined using fluorescence microscopy.

Interpreting the results of indirect immunofluorescence

The immunofluorescent slides are examined to determine the presence of autoantibodies via the patterns of immune deposition. The results are subjective and indirect immunofluorescence cannot be used reliably to monitor disease severity and/or its treatment.

Different staining patterns

Intercellular staining (ICS) pattern

Basement membrane zone (BMZ) pattern

Immunofluorescent patterns in skin diseases

Sensitivity of a diagnostic tool refers to its ability to accurately identify those with the disease (true positive).

Specificity is its ability to accurately identify those without the disease (true negative).

Occasionally the test can produce false results.

The result may be negative even if the patient has the disease (false negative).

The result may be positive in a patient without the disease (false positive).